The HIV-1-encoded Tat protein is a potent transcriptional activator that is essential for viral replication. The long-term objective of this proposal is to gain a better understanding of the control of HIV-1 gene expression by Tat and cellular factors at the molecular level. This study will focus on a novel RNA Pol II complex (Tat- SF) that efficiently mediates Tat-enhanced transcription (Tat function). Tat-SF contains P-TEFb, Spt5/Spt4, and Tat-SF1 and novel polypeptides, but none of the yeast coactivator SRB/MED-like proteins found associated with RNA Pol II holoenzyme. Notably, these Tat-SF cofactors can jointly complement the transcription initiation competent RNA Pol II holoenzyme for potent transcription elongation, reinitiation, and Tat transactivation, indicating that Tat-SF contains critical transcription factors. The mechanisms by which Tat-SF and RNA Pol II holoenzyme collectively mediate potent Tat-enhanced HIV-1 transcription are poorly understood. This proposal has three specific aims. The first will determine and compare the polypeptide composition of Tat-SF and Tat-SF cofactors derived from HeLa cytoplasm, nuclear extract, and nuclear pellet (chromatin). The second aim will examine the mechanisms by which Tat-SF cofactors mediate potent Tat-enhanced HIV-1 transcription. These studies will provide insights into synergisms of Tat-SF cofactors within themselves and with other transcription factors and protein modifications (e.g. phosphorylation, acetylation, methylation) for potent Tat activation. The third aim will specifically study the interplay of Tat-SF and the SRB/MED complex for efficient general transcription reinitiation and elongation. These experiments will include the detailed characterization of a potential SRB/MED complex-containing scaffold formed on the HIV- 1 promoter that could be utilized by Tat-SF for efficient transcription. The effects of Tat on Tat-SF- and RNA Pol II holoenzyme-mediated transcription elongation and re-initiation will also be examined. Collectively, these studies will identify novel factors and mechanisms that are important for single and multiple round of HIV-1 transcription. They will also establish a unique well-defined in vitro system for future elaboration of the link between HIV-1 transcription elongation and mRNA processing either on naked or chromatin DNA templates. Together, understanding Tat-enhanced HIV-1 transcription at the molecular level will be instrumental for future therapeutic antiviral strategies to block viral replication.